We and others have recently investigated patients with type 1 diabetes for the frequency and function of a population of regulatory T cells (Treg), characterized by the simultaneous expression of CD4 and CD25. Our studies did not support the notion that altered CD4+CD25+ T cell frequencies are associated with type 1 diabetes, but rather identified type 1 diabetes related alterations in the functional activities of these cells in terms of suppressing effector T (Teff) cell responses in vitro. The need to bring resolution to the aforementioned published discrepancies in frequency and function of Treg in type 1 diabetes, as well as investigate the potential for Teff cell defects, would be afforded with expanded studies that include the parameters of age, metabolic control, and disease duration, as well as to define (in association with Projects 1 and 2) the cellular and molecular mechanism(s) underlying this defect. Therefore, the overall objective of Project 3 is to improve our understanding the mechanisms of immune regulation afforded by CD4+CD25+ T cells, identify their contribution to the pathogenesis of type 1 diabetes, and evaluate the potential of these cells to serve as a marker for autoimmune disease activity. Our specific aims are designed to test the hypothesis that Treg cells are functionally defective in type 1 diabetes, as a result of dysregulated interactions with APC, Teff, and NKT cells, and that the cellular &molecular basis for this defect resides in pathways controlling the phenotypic signature of Treg including surface CD25, FOXP3, as well as TGFfS. This hypothesis has been formed based on our observations of deficient functional activities of Treg in human type 1 diabetes, recent data suggesting the surface expression/stability of CD25 is crucial to maintaining regulatory homeostasis between Treg and Teff, literature indicating the immunological synapse with other immune system cells (e.g., DC, NKT cells) may influence the functional activities of Treg, as well as information suggesting key roles for a limited number of cytokines (e.g., IL-2, IL-10, TGFp) and matrix metalloproteinases are associated with these regulatory processes. The Project has two specific aims: 1) Identify the influence of age, type 1 diabetes, and metabolic control on the frequency and function of regulatory T cells defined by co-expression of CD4 and CD25, as well as on the cellular expression of the fork-head transcription factor FoxPS. 2) Define the molecular mechanisms underlying deficiencies in CD4+CD25+ T cell function in subjects with type 1 diabetes. The successful completion of these studies could provide key information to fill an existing knowledge void regarding the mechanistic interactions between specific cell populations that underlie the failure of immune regulation which results in type 1 diabetes.